The dynamic instability of microtubules

As already described, maintenance of genomic integrity is dependent on the properly assembled mitotic spindle. MTs form the mitotic spindle. They are divided into the following subsets: astral MTs ensure the spindle interactions with the cell cortex; kinetochore MTs tether the chromosomes to the mitotic spindle; interpolar MTs interdigitate between the spindle poles forming an array of antiparallel MTs named the “spindle midzone” (Fraschini, 2016).

Each MT consists of thirteen protofilaments arranged in a circle, and each protofila-ment is a polymer ofαβ-tubulin dimers assembled in a head-to-tail fashion (Fraschini, 2016). As a result of the directionality in the organisation of each protofilament,

Figure 1.15: Microtubule dynamic in-stability.The MT growth (polymerisation) and shrinkage (depolymerisation) are driven by hy-drolysis of the GTP bound to β-tubulin. New GTP-bound αβ-tubulin dimers are incorporated into growing MTs by hydrolysing the GTP which is already incorporated in the MT (at the tip), thus keeping the GTP intact. Once this GTP-cap is lost, due to the intrinsic instability of GDP-bound dimers, the MT growth will be rapidly switched to the shrinkage – a catastrophe. The rescue, which is a switch from shrinkage to growth, is possibly explained by the presence of “GTP islands” or

“rescue factors” in the MT lattice. Adapted from (Akhmanova & Steinmetz, 2015).

the ends of MTs acquire distinct proper-ties. One of the ends (β-tubulin) is cha-racterised by fast polymerisation, also de-scribed as growing properties, and there-fore, is called the plus-end (or plus-tip).

The opposite end (α-tubulin) is slow growing and is called the minus-end (or minus-tip).

The exquisite property of MTs is their ability of a fast switch between growth and shrinkage (depolymerisa-tion), described as dynamic instability (Figure 1.15; Mitchison & Kirschner, 1984). The switch from growth to shrinkage is denoted as a catastrophe, whereas the opposite way is called res-cue (Gardner, Zanic, & Howard, 2013).

The GTP-cap model explains the cur-rent understanding of dynamic instabil-ity. The growing of the MTs is an

energy-consuming process. The addition of each new αβ-dimer is driven by hydrolysis of a GTP molecule bound to β-tubulin at the plus-end. It is generally accepted that the stability of the newly formed MT tip is achieved by a so-called GTP cap, which contains the conformationally more stable GTP-tubulin, whereas the MT shaft contains the intrinsically unstable GDP-tubulin that is the MT grows as long as the GTP cap is present (Akhmanova & Steinmetz, 2015).

The dynamic instability of MTs is essential for the proper functioning of the mitotic spindle. First, changes in the growth state itself create pulling and pushing forces, indispensable for the positioning and orienting the mitotic spindle (Grill &

Hyman, 2005; McNally, 2013; Toli´c-Nørrelykke, 2008; H.-Y. Wu, Nazockdast, Shelley,

& Needleman, 2017). Moreover, different MT-interacting proteins bind the ends of MTs with different affinity, which, for instance, contributes to the directional movement of MT-bound motor proteins (Goodson & Jonasson, 2018). The movement of MT-bound motor proteins generates forces that allow adjacent MTs to slide in relation to one another – a feature important for bipolar mitotic spindle formation and segregation of chromosomes (Fraschini, 2016). Last but not least, the dynamic instability underlies the “search and capture” model described above (Heald &

Khodjakov, 2015).

The switch between rescue and catastrophe events is a strictly regulated process.

As depicted in Figure 1.16, a long list of proteins, including MAPs (microtubule-associated proteins), +TIPs (plus-end-tracking proteins), -TIPs (minus-end-tracking proteins), are involved in the control of MT dynamics. Moreover, the activity of these proteins is tightly coordinated with cell cycle progression. An example of +TIPs is the family of MT polymerases XMAP215 (Xenopus microtubule-associated protein), which recruits and incorporates αβ-dimers in the plus-end of the MT (Brouhard et al., 2008). Therefore, misbalance in the activity of the members of this family can result in errors during mitosis. For instance, the MT polymerase ch-TOG (colonic and hepatic tumour overexpressed gene protein, also termed CKAP5) has been shown to be involved in the regulation of kinetochore-MT attachment stability, and changes in its activity can potentially lead to the formation of lagging chromosomes (Cheeseman, Harry, McAinsh, Prior, & Royle, 2013; Miller, Asbury, & Biggins, 2016).

The end-binding proteins (EBs), which belong to +TIPs can moderately increase

Figure 1.16: The complexity of regulation of microtubule dynamics. The diagram maps only some signalling pathways and the crosstalk between them, however, it can already be appreciated how strict and redundant the control of MT dynamics is. In all cases, the entire process is narrowed down to the modulation of the polymerisation of MTs via recruiting rescue factors, changing the activity of XMAP215 MT polymerase or MCAK MT depolymerase. Also, involvement of cell cycle kinases, e.g. Aurora B and CDK1, is shown. Illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).

MT polymerisation rates in vitro, possibly by influencing the stability of MT ends (Akhmanova & Steinmetz, 2015; Lansbergen & Akhmanova, 2006).

MT depolymerisation, or shrinkage, is regulated by the kinesin family of MT depolymerases, which are kinesin-13, kinesin-8 or kinesin-14 (Akhmanova & Stein-metz, 2015; Walczak, Gayek, & Ohi, 2013). Likewise, changes in the activity of these proteins are prone to generating errors during mitosis. Depletion of the kinesin-8 KIF18A attenuates chromosome movements and delays the anaphase onset (Mayr et al., 2007; Stumpff, von Dassow, Wagenbach, Asbury, & Wordeman, 2008; Stumpff, Wagenbach, Franck, Asbury, & Wordeman, 2012). A member of the kinesin-13 family MCAK (mitotic centromere-associated kinesin) has been shown to be implicated in the alignment of chromosomes at metaphase (Illingworth, Pirmadjid, Serhal, Howe,

& Fitzharris, 2010) and sister chromatid segregation (Maney, Hunter, Wagenbach,

& Wordeman, 1998). Moreover, it has been shown that the major players of cell cycle Aurora B-PLK1 signalling regulates MCAK at kinetochores, thus ensuring the prompt correction of erroneous kinetochore-MT attachments and preventing formation of lagging chromosomes during anaphase (Shao et al., 2015).